Method for inhibiting yeast growth

ABSTRACT

The invention relates to the use of microbes  Lactobacillus rhamnosus  LGG, ATCC 53103,  Latobacillus rhamnosus  LC705, DSM 7061, and  Propionibacterium freudenreichii  ssp.  shermanii  PJS, DSM 7067 in inhibiting yeast growth, for preventing and treating diseases caused by yeast and for relieving yeast-related symptoms in animals or humans.

FIELD OF THE INVENTION

The invention relates to inhibiting the growth of yeasts. In particular,products and methods for inhibiting the growth of yeasts and forpreventing and treating diseases caused by yeasts are disclosed.

BACKGROUND OF THE INVENTION

Yeasts are constantly present in the living environment and the organsystem of humans. Even a healthy individual has Candida albicans yeastgrowing on the mucosal membrane and in the entire area of thegastroin-testinal tract (Shay K, Truhlar M R, Renner R P. Oropharyngealcandidosis in the older people. J Am Geriatr Soc 1997;45:863-870). Theoral cavity and the tissue between teeth also provide an excellentgrowth medium for a number of species of microbes, among which can befound for example the Candida albicans species and, to a lesser amount,C. glabrata and C. tropicalis yeasts. Normally yeast cells are in apassive state and their growth does not harm healthy individuals. Inunfavourable conditions yeasts, such as C. albicans, begin to formhyphae and thereby penetrate deeper into the mucosal membranes. Thisresults in a local yeast infection, which in the mouth appears forexample as oral candidiasis, stomatitis, or glossitis.

Yeast infections are usually preceded by reduced resistance, which maybe caused by certain medication, for example use of broad-spectrumantibiotics, corticosteroids or cytostatics, by diabetes, malign tumors,or immunodeficiency. Micro-organisms of the mouth spread easily, carriedby blood circulation, to other parts of the organ system, which may havesevere consequences, such as septicemia, endocarditis and meningitis,particularly in persons whose general state of health has deteriorated(Shay et al. 1997).

The most common and most important cause of yeast infections in humansis Candida albicans (Mäkelä et al. 1988. L{umlaut over(aa)}ketieteellinen mikrobiologia (Medical microbiology), 5^(th) revisededition, pp. 270-271. Published by Kustannus Oy Duodecim 1988). C.albicans can be found relatively often in the gastrointestinal tract ofhealthy humans: 30 to 50% carry it in the mouth and about 1% on healthyskin and in the urinary tract. Also other species of the Candida genuscan be found occasionally, the most important ones being: C. tropicalis,C. pseudotropicalis, C. parapsilosis, C. knisei, and C. guilliermondi,which act as opportunistic pathogens in humans, similarly as C. albicans(Mäkelä et al. 1988).

Elderly people typically suffer from a number of diseases, which,together with the medication used for treating them, may weaken theirimmunity and, at the same time, their dental health (Pajukoski H,Meurman J H, Snellman-Gröhn S, Sulkava R. Oral health in hospitalizedand nonhospitalized community-dwelling elderly patients. Oral Surg OralMed Oral Pathol Oral Radiol Endod 1999:88:437443). In addition, poordental hygiene submits elderly people to yeast infections(Budtz-Jorgensen E, Mojon P, Banon-Clement J M, Baehni P. Oralcandidosis in long-term hospital care: comparison of edentulous anddentate subjects. Oral Dis 1996;2:285-290). Ageing as such increases theappearance of yeast and the amount of it, which implies that the abilityof the organ system to inhibit yeast growth reduces with age (Lockhart SR, Joly S, Vargas K, Swails-Wenger J, Enger L, Soil D R. Naturaldefenses against Candida colonization breakdown in the oral cavities ofthe elderly. J Dent Res 1999;78:857-868).

In elderly people living in Helsinki, Finland, yeast growth was found in75%, high contents being found in 33% (Närhi T O, Ainamo A, Meurman J H.Salivary yeasts, saliva, and oral mucosa in the elderly. J Dent Res1993;72:1009-1014). Candida yeast infection, in turn, has been found in60% of those who carry yeast (Wilkieson C, Samaranayake L P, MacFarlaneT W, Larney P J, etc. Oral candidosis in the elderly in long termhospital care. J Oral Pathol Med 1991;20:13-16). The most common yeastsafter Candida albicans are C. glabrata (29%), C. tropicalis (13%),Saccharomyces cerevisiae (11%), and C. parapsilosis 89%) (Lockhart etal. 1999).

One important factor increasing oral yeast growth is reduced salivation(Närhi et al. 1993). A five-year follow-up study of the effects ofageing showed that stimulated secretion of total saliva reduces withageing, whereas buffer capacity increases (Närhi T O, Kurki N, Ainamo A.Saliva, salivary micro-organisms, and oral health in the home-dwellingold elderly—a five-year longitudinal study. J Dent Res1999;78:1640-1646). Endocrinological diseases and abundant use ofmedication in particular reduce salivation (Pajukoski H, Meurman J H,Snellman-Gröhn S. Keinänen S, Sulkava R. Salivary flow and compositionin elderly patients referred to an acute care geriatric ward. Oral SurgOral Med Oral Pathol Oral Radiol Endod 1997; 84: 265-71). Yeast contentsare clearly higher for those whose rate of salivation and buffercapacity has reduced (Närhi et al. 1993). The rate of salivation ofelderly Finns varies between 0.6 and 1.0 ml/mm (Pajukoski et al. 1997);reduced secretion (<0.7 ml/mm) is found with as much as 55% and lowbuffer capacity with 34% of the elderly (Pajukoski et al. 1997).

Candidosis has conventionally been treated with antifungal agents, suchas nystatin, and more severe cases with fluconazole or itraconazole(Shay et al. 1997). Also more efficient oral hygiene has enabled toreduce Candida colonization in the oral mucosal membranes of elderlyconfined in long-term care (Budtz-Jorgensen E, Mojon P, Rentsch A,Deslauriers N. Effects of an oral health program on the occurrence oforal candidosis on a longterm care facility. Community Dent OralEpidemiol 2000;28:141-149). The use of chlorhexidine/xylitol chewing-gumalso enabled the amount of yeast to be reduced by 22% (Simons D, Kidd EA M, Beighton D, Jones B. The effect of chlorhexidine/xylitolchewing-gum on cariogenic salivary microflora: A clinical trial inelderly patients. Caries Res 1997;31 :91-96).

Certain groups, such as patients in long-term care and elderly people,are also prone to general infections due to reduced immunity,malnutrition and chronic diseases. In addition, in elderly people oralhygiene and oral microbial flora have been found to be associated withinfections of the lower respiratory ducts, such as pneumonia andbronchitis (see Scannapieco F A. Role of oral bacteria in respiratoryinfection. J Petiodontol 1999;70:793-802). An infection is presumablycaused when pathogenic microbes are transferred through aspiration fromthe mouth to the respiratory ducts. The most common infections affectingpatients in long-term care are: infections of the upper and lowerrespiratory ducts (70%), infections of the urinary tract (12%),gastroenteritis and diarrhea (12%) as well as dermatitis and infectionsof the soft tissue (6%) (Orr P H, Nicolle L E, Duckworth H, Brunka J,Kennedy J, Murray D et al. Febrile urinary infection in theinstitutionalized elderly. Am J Med 1996;100:71-77). Hygieneintervention has enabled to reduce infections of the upper respiratoryducts of elderly people in long-term hospital care by 16% (Makris A T,Morgan L, Gaber D J, Richter A, Rubino J R, Effect of comprehensiveinfection control program on the incidence of infections in long-termcare facilities. Am J Infect Control 2000;28:3-7).

Another main area where yeast appears is the genital area; in women inparticular yeast infections are common. The most common gynecologicalsymptom is vaginitis; it is also one of the main symptoms of patientswho seek medical consultation (Makela et al. 1988). Vaginitis is mostcommonly caused by a bacterium, the second most common cause being ayeast fungus, C. albicans and C. glabrata being the species that appearmost often. Candida albicans is a yeast that belongs to the normalmicroflora of the vagina, and normally it does not cause infections.Usually the bacterial flora of the vagina restricts yeast growth, but incertain conditions yeast growth becomes excessive. Risk factors for ayeast infection include use of antibiotics (particularly broad-spectrumantibiotics), pregnancy, diabetes, diseases that cause immunedeficiency, and use of corticosteroids. Yeast fungus can be found in 10to 20% of patients at gynaecological outpatient departments, but onlysome of them become affected by a clinical vulvovaginitis caused byyeast. A basic pH favours yeast growth. The virulence of yeast dependson the yeast content, the invasiveness of its mycelium, its steroidreceptors, and on the ability of yeast to form proteases, for example.Research has shown that fungal hyphae are capable of penetrating thevaginal epithelium (Mäkelä et al. 1988).

Lactic acid bacteria have been used with varying results for preventingvaginitis. Yoghurt intake for 6 months reduced Candida colonization andthe occurrences of vaginitis (Hilton et al. 1992. Ingestion of yoghurtcontaining Lactobacillus acidophilus as prophylaxis for Candidalvaginitis. Ann Intern Med 1992;116:353-357). On the other hand,according to the research carried out by the Shalev group (1996)Lactobacillus acidophilus yoghurt, in comparison with pasteurizedyoghurt, reduced vaginitis caused by bacteria, but not that caused byCandida yeast (Shalev et al. 1996. Ingestion of yoghurt containingLactobacillus acidophilus compared with pasteurized yoghurt asprophylaxis for recurrent Candidal vaginitis and bacterial vaginosis.Arch Fam Med 1996;5.593-596; Sieber R, Dietz U-T. Lactobacillusacidophilus and yogurt in the prevention and therapy of bacterialvaginosis. Int Dairy J 1998; 8:599-607; see also Redondo-Lopez et al.1990. Emerging role of lactobacilli in the control and maintenance ofthe vaginal bacterial microflora. Rev Infect Dis 1990;12:856-872).

Yeast fungus also causes urethritis.

Several studies have shown that 20-40% of people carry C. albicans inthe gastrointestinal tract (Lennette et al. 1985. Manual of clinicalmicrobiology, 4^(th) edition. American Society for Microbiology,Washington D.C., 1985). Overgrowth of yeast in the gastrointestinaltract manifests itself usually as diarrhea.

The term ‘yeast syndrome’ is usually used to refer to increased growthof Candida albicans yeast in the gastrointestinal tract, which isconsidered to inhibit the immune system and to cause differentsyndromes. Yeast overgrowth has been associated with a number ofsystemic symptoms, such as symptoms of the central nervous system,different aches, fatigue and symptoms of the intestines. It is assumedthat the symptoms are due to toxins released by yeasts. However, thereis no scientific evidence of a connection between such symptoms andyeast. Yeast syndrome is treated with a low-carbohydrate, yeast-freediet, possibly favouring soured and/or fibre-rich nutrients. Souredfoods and soured dairy products in particular form an important elementof the diet treatment. Consequently, lactic acid bacteria are often usedto balance a disturbed intestinal flora.

Yeast thus causes many kinds of diseases, both indirectly and directly.There is therefore a constant need to find new means for inhibitingyeast growth and activity, for preventing and treating diseases causedby yeast, and for relieving yeast-related symptoms.

As an alternative to medical treatment, or in addition to it, attemptsare nowadays being made to utilize other health care means as well. Oneof the most recent methods is to use health-promoting nutrients ornatural products, which have in fact been warmly welcomed by consumers.Health-promoting products inhibiting yeast growth and activity wouldtherefore be a highly welcome addition to the range of commerciallyavailable products. The products should preferably be pleasing andsomehow familiar to the consumer, which would make them easy to adopt asa part of the normal daily diet, for example.

The effects of probiotics as anti-microbial agents have been describedin the literature of the art. Lactic acid bacteria are capable ofproducing anti-microbial compounds, organic acids, lactic acid, fattyacids, hydrogen peroxide, diacetyl, carbon dioxide, and bacteriocins,which enables them to inhibit the growth of pathogenic microbes(McGroarty J A. Probiotic use of lactobacilli in the human femaleurogenital tract. FEMS Immunol Med Microbiol 1993;6:251-264). Somestrains of Lactobacillus acidophilus prevented the growth of Candida invitro by producing hydrogen peroxide (Jack M, Wood J B, Berry D R.Evidence for the involvement of thiocyanate in the inhibition of Candidaalbicans by Lactobacillus acidophilus. Microbios 1990;62:3746;Fitzsimmons N, Berry D R. Inhibition of Candida albicans byLactobacillus acidophilus: evidence for the involvement of a peroxidasesystem. Microbios 1994;80:125-133). It has been discovered thatLactobacillus rhamnosus LGG ATCC 53103 also produces an anti-micronialcompound, possibly a short-chain fatty acid, that inhibits the in vitrogrowth of Escheichia coli, Pseudomonas, Salmonella, Streptococcus,Bacillus, Clostridium, and Bifidobacterium, for example (Silva M,Jacobus N V, Deneke C, Gorbach S L. Antimicrobial substance from a humanLactobacillus strain. Antimicrobial Agents Chemother 1987;31:1231-1233).

Lactic acid bacteria may also prevent the adhesion of other microbes toepithelial cells. For example, some Lactobacillus acidophilus and L.casei strains have been found to prevent 22-46% of the adhesion of C.albicans to uroepithelial cells in in vitro conditions (Reid G, TieszerC, Lam D. Influence of lactobacilli on the adhesion of Staphylococcusaureus, and Candida albicans to fibers and epithelial cells. J IndustMicrobiol 1995;15:248-253). In animal tests Lactobacillus rhamnosus LGGhas been discovered to reduce the amount of C. albicans in thegastrointestinal tract of mice and candidosis in the mouth area bystimulating a cell-mediated immune response to antibodies of C.albicans. (Wagner R D, Pierson C, Warner T, Dohnalek M, Farmer J.Roberts L et al. Biotherapeutic effects of probiouic bacteria oncandidiasis in immunodeficient mice. Infect Immun 1997;65:41654172;Wagner R D, Pierson C, Warner T. Dohnalek M, Hilty M, Balish E.Probiotic effects of feeding heat-killed Lactobacillus acidophilus andLactobacillus casei to Candida albicans—colonized immunodeficient mice.J Food Protect 2000;63 :638-644).

When acting together, Lactobacillus LC 705 and Propionibacteriumfreudenreichii ssp. shermanii PJS have been discovered to inhibit yeastgrowth in yoghurts and curd cheese (Suomalainen T, Mäyrä-Mäkinen A.Propionic acid bacteria as protective cultures in fermented milks andbreads. Lait 1999;79:165-174).

Lactic acid bacteria and their cellular structures may activate theresistance of the organ system by increasing the activity of macrophagesand natural killer cells, the amount of T and B cells and the proportionof antibodies (Perdigon G, Alvarez s, Rachid M, Agüero G, Gobbato N.Symposium: Probiotic bacteria for humans: Clinical systems forevaluation of effectiveness. Immune system stimulation by probiotics. JDairy Sci 1995;78:1597-1606).

Lactobacillus rhamnosus LGG has also been found to enhance the normalresistance of the intestines against harmful bacteria, viruses, andyeasts (Kaila M, Isolauri E, Soppi E et al. Enchancement of thecirculating antibody secreting cell response in human diarrhea by ahuman Lactobacillus strain. Pediatr Res 1992;32:141-144; Wagner et al.1997). In animal tests Lactobacillus GG has also increased the contentof secretory IgA in saliva (Negretti F, Casetta P, Clerici-Bagozzi D,Marini A. Researches on the intestinal and systemic immunoresponsesafter oral treatments with Lactobacillus GG in rabbit. Phisiopath Clin1997;7:15-21). The secretory IgA of the mucosal membrane is known toprotect the respiratory ducts, gastrointestinal tract, and urogenitalorgans against infections (Nagura H. Mucosal defence mechanism andsecretory IgA system. Bifidobacteria Microflora 1990;9: 17-25). Lacticacid bacteria may thus also reduce infections of the respiratory tractand the gastrointestinal tract, indications of which have already beenobtained in studies conducted among day-care children (Hatakka K,Savilahti F, Pönkä A, Meurman J H, Poussa T, Näse L, Saxelin M, KorpelaR. The effect of long-term consumption of a probiotic milk on theinfections of children attending day care centres: a double-blindrandomised trial. Submitted to BMJ in May, 2000).

BRIEF DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide means enabling thegrowth and activity of yeast in humans and in animals to be inhibited orreduced. This is achieved by the product, use and method of theinvention, which are characterized by what is stated in the independentclaims. Preferred embodiments of the invention are disclosed in thedependent claims.

The present invention is based on using specific probiotics forinhibiting yeast growth and activity, for preventing and treatingdiseases caused by yeast, and for relieving yeast-related symptoms inhumans and in animals.

The invention thus relates to the use of the microbes Lactobacillusrhamnosus LGG, ATCC 53103, Lactobacillus rhamnosus LC705, DSM 7061, andPropionibacterum freudenreichii ssp. shermanii PJS, DSM 7067 forinhibiting yeast in humans and in animals.

For the inventive purpose the bacteria may be consumed separately or ina combination. They can be consumed as such, for example in the form ofa lyophilized product, or used as an additive or an ingredient of edibleproducts, such as dairy products and drinks. For the preparation of acombination, mixed cultures or pure cultures of each bacterium can beused. The combination can also be prepared in a unit dosage form, suchas a capsule. The capsule may contain all the above bacteria, preferablyin the form of lyophilized cultures, or a series of three capsules maybe prepared, one for each bacterium.

The invention thus also relates to the use of the bacteria Lactobacillusrhamnosus LGG, ATCC 53103, Lactobacillus casei ssp. rhamnosus LC705, DSM7061, and Propionibacterium freudenreichii ssp. shermanii PJS, DSM 7067for preparing a product for inhibiting yeast.

The product can be a food industry product or pharmaceutical industryproduct, for example, or a health-promoting or a natural product.Preferred products include health-promoting dairy products, such as acheese, into which the microbes are added in connection with themanufacture of the product. The microbes may also act as starters and aselements forming the structure of the cheese or other product. A secondpreferred product group includes pharmaceutical preparations,particularly tablets and capsules, that contain auxiliary agents andadditives commonly used in these products, and possibly also otheractive ingredients, in addition to the above micro-organisms.Particularly preferred products include preparations for oralconsumption, such as tablets and capsules, containing xylitol, inaddition to LGG, LC705, and PJS. Apart from the microbes used accordingto the invention, the products may contain other microbes as well. Thebacteria, combinations and other products disclosed herein have aneffect on the growth and activity of yeast appearing in the human organsystem and they prevent yeast infections. They are thus useful forpreventing disorders and diseases caused by yeast, for relievingsymptoms related thereto, and for general health improvement.

The invention further relates to a method for inhibiting yeast growthand for preventing or treating diseases caused by yeast or for relievingyeast-related symptoms in humans and in animals, the method comprisingadministering the microbes Lactobacillus rhamnosus LGG, ATCC 53103,Lactobacillus casel ssp. rhamnosus LC705, DSM 7061, andPropionibacterium freudenreichii ssp. shermanii PJS, DSM 7067 to anindividual in need thereof in an amount sufficient to produce thedesired result.

DETAILED DESCRIPTION OF THE INVENTION

The present invention thus relates to the use of specific probiotics forinhibiting yeast growth and activity in the organ system of humans oranimals.

Probiotics are live microbes that, when administered to humans oranimals, promote the health of the host by improving the microbialbalance in the intestines; in this way, or in addition to this,probiotics may have many other useful properties as well.

The most important probiotics are lactic acid bacteria, propionic acidbacteria and bifido bacteria. These belong inherently to the organsystem of humans and animals. Lactobacilli are an important part of thenormal bacterial flora of human organ system (Redondo-Lopez V, Cook R L,Sobel J D. Emerging role of lactobacilli in the control and maintenanceof the vaginal bacterial microflora. Rev Infect Dis 1990;12:856-872).Propionic bacteria, in turn, appear on the skin and in thegastrointestinal tract (MacFarlane G T, Allison C, Gibson S A W,Cummings J H. Contribution of the microflora to proteolysis in the humanlarge intestine. J Appl Bacteriol 1988;64:37-46). Due to their safetyand health-promoting effects, probiotics are often used in foodstuffs aswell.

The strains Lactobacillus rhamnosus LGG, ATCC 53103, Lactobacillus caseissp. rhamnosus LC705, DSM 7061, and Propionibacterum freudenreichii ssp.shermanii PJS, DSM 7067 to be used in the invention have been describedin the prior art.

Lactobacillus rhamnosus GG (LGG) has been described for example in U.S.Pat. No. 5,032,399, Gorbach & Goldin. The strain has been isolated fromhuman feces, it is able to grow well in pH 3 and survives even lower pHvalues as well as high bile acid contents. The strain exhibits excellentadhesion to both mucus and epithelial cells. Lactic acid yield fromglucose is good: when grown in MRS broth, the strain produces 1.5-2% oflactic acid. The strain does not ferment lactose. The strain employs thefollowing carbohydrates: D-arabinose, ribose, galactose, D-glucose,D-fructose, D-mannose, rhamnose, dulcitol, inositol, mannitol, sorbitol,N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, cellobiose,maltose, saccharose (slowly), trehalose, melezitose, gentibiose,D-tagatose, L-fucose, and gluconate. The strain grows well at +15-45°C., the optimum temperature being 30-37° C. Lactobacillus rhamnosus GGis deposited with the depository authority American Type CultureCollection under accession number ATCC 53103.

Lactobacillus rhamnosus GG is a natural bacterial strain in humans andits probiotic effects have been widely studied (Saxelin M. LactobacillusGG—a human probiotic strain with thorough clinical documentation. FoodRev Int 1997;13:293-313). It remains viable in the gastrointestinaltract and is capable of temporarily colonizing the intestines (Goldin BR, Gorbach S L, Saxelin M, Barakat S, Gualtieri L, Salminen S. Survivalof Lactobacillus species (strain GG) in human gastrointestinal tract.Dig Dis Sci 1992;37:121-128). LGG seems to be capable of colonizing, atleast temporarily, the oral cavity as well, because the bacterium wasfound in the test persons' saliva for as long as two weeks after aseven-day period of LGG yoghurt consumption had ended (Meurman J H,Antila H, Salminen S. Recovery of Lactobacillus strain GG (ATCC 53103)from saliva of healthy volunteers after consumption of yoghurt preparedwith the bacterium. Microbiol Ecol Health Dis 1994;7:295-298). LGG iscurrently added to a number of commercially available sour milk andjuice products (Gefilus®).

Lactobacillus casei ssp. rhamnosus LC705 is described in greater detailin Fl Patent 92498, Valio Oy. LC705 is a gram-positive short rodoccurring in chains; it is homofermentative; weakly proteolytic; growswell at +15-45° C.; does not produce ammonia from arginine; iscatalase-negative; when grown in MRS broth (LAB M), the strain produceslactic acid (1.6%) having the optical activity of the L(+)configuration; the strain decomposes citrate (0.169%), thereby producingdiacetyl and acetoin; the strain ferments at least the followingcarbohydrates (sugars, sugar alcohols): ribose, galactose, D-glucose,D-fructose, D-mannose, L-sorbose, rhamnose, mannitol, sorbitol,methyl-D-glucoside, N-acetylglucosamine, amygdalin, arbutin, esculin,salicin, cellobiose, maltose, lactose, sucrose, trehalose, melezitose,gentiobiose, D-turanose and D-tagatose. LC705 adheres weakly to mucuscells, but moderately to epithelial cells. The viability of the strainis good in low pH values and high bile acid contents. The strainsurvives well a salinity of 5% and fairly well a salinity of 10%.Lactobacillus casei ssp. rhamnosus LC705 is deposited with the DeutscheSammiung von Mikroorganismen und Zellkulturen GmbH (DSM) under accessionnumber DSM 7061.

Lactobacillus rhamnosus LC705 is used for example in the manufacture ofEmmental cheese to prevent butyric acid fermentation caused byclostridia. The strain is also used in foodstuffs where it functions asan inhibitor of yeast and mold growth. In biological preservation LC705strain is combined with Propionibacterium freudenreichii ssp. shermaniiPJS (Fl 92498).

Propionibacterium freudenreichii ssp. shermanii JS (PJS) is alsodescribed in greater detail in Fl Patent 92498, Valio Oy. PJS is agram-positive short rod; it ferments glucose, fructose, galactose andlactose; it ferments well lactate; and its optimum growth temperature is32° C. The viability of the strain in low pH values and high bile acidcontents is excellent. Propionibacterium freudenreichii ssp. shermaniiJS is deposited with the Deutsche Sammiung von Mikroorganismen undZelikulturen GmbH (DSM) under accession number DSM 7067.

In addition to the microbes to be used in accordance with the invention,the products to be manufactured may also contain other microorganisms,such as micro-organisms and probiotics contained in starters used in thedairy industry. There are numeral well-documented strains of startersthat are commercially available from producers such as Hansen A/S,Denmark, and Danisco/Niesby GmbH, Germany.

The micro-organisms to be used according to the invention are cultivatedusing conventional methods, either as pure cultures or as differentmixed cultures. The cultures can be used as such, or they can beprocessed as desired, for example purified, concentrated, lyophilized orfinished to produce different products. The preparation of themicro-organisms to be used according to the invention is described indetail for example in publications Fl 92498 and Fl 20010157.

According to the invention, a sufficient amount of probiotics is used toproduce the desired yeast inhibiting effect. The amount of eachindividual probiotic may therefore vary within a large range dependingon for example the total amount of probiotics cells, the total dailydose and on other properties and ingredients of the product. Theprobiotics content in a daily dose of a combination is usually about10⁶-10¹⁰ cfu.

According to the invention, the probiotics are suitable for consumptionas such or formulated as capsules, pills, or tablets, for example, inprocesses conventionally applied for preparing pharmaceutical products.The probiotics to be used according to the invention can also be addedto different edible products, such as foodstuffs, products of thebeverage and confectionary industries, to health-promoting products,natural products, etc. Within the scope of the present invention, dairyproducts containing the specific probiotics, particularly cheeses andspreads, yoghurts and other sour milk products, and children's foods,juices and soups, as well as capsules, pills, and tablets are consideredas preferred embodiments.

The end products are produced using conventional methods, the probioticsbeing added either during the process of preparing the product orafterwards, during the finishing.

The tests, which are described in greater detail in the publication,were carried out to study the effects of the probiotics to be usedaccording to the invention on inhibiting oral yeast growth. The mostcommon one of these yeasts, Candida albicans, which is a goodrepresentative of yeast species and widely present in the humanorganism, is most useful also as a model organism for other yeastspecies appearing in the mouth and in other parts of the organ system.The results presented in the examples show that the probiotics usedaccording to the invention have a statistically significant reducingeffect on the amount of oral yeasts. According to preliminary results itis evident that the invention is applicable in inhibiting, reducing andslowing down to a wide extent the growth and activity of yeast appearingalso in other parts of the organ system, such as the intestinal tractand the urogenital area.

The invention will be described in detail with reference to thefollowing examples, which are provided only to illustrate the inventionand are not to be considered to restrict its scope of protection in anyway.

EXAMPLE 1

The Effect of Probiotics on the Occurrence of Candida albicans

The main purpose of the study was to find out whether a probioticcombination containing lacto bacilli and propionic acid bacteria couldbe used for reducing the occurrence of Candida albicans in the mouth.Emmental-type cheese containing live Lactobacillus rhamnosus LGG, ATCC53103, Lactobacillus rhamnosus LC705, DSM 7061, and Propionibacteriumfreudenreichii ssp. shermanii PJS, DSM 7067 microbes was selected as thetest material. Cheese was chosen because it is a product that belongs toan ordinary diet, it is pleasant to eat and easy to portion out inaccordance with the study purpose. The cheese used as a reference wasEdam cheese that did not contain the three bacterial strains in questionbut normal starter microbes, which were lactococci.

240 people were recruited for the study, their ages varying from 70 to100 years.

The study was carried out as a placebo-controlled double blind test,with parallel study groups. The total study period was 19 weeks, with arun-in period of 3 weeks and 16 weeks of intervention. Use of foodstuffscontaining probiotic bacteria was forbidden during the entire study. Thelist of forbidden foodstuffs contained e.g. Emmental and Polar cheeses,curd cheese, sour milk products containing live lactic acid bacteria,probiotics juices and different capsules and similar compressedproducts.

During the entire study period, the test persons adhered to their usualhabits regarding oral hygiene and their normal ways of living.

Intervention: The cheese intervention continued for 16 weeks (wk).During this time half of the test persons consumed 50 g (=6-7 slices) ofcheese containing probiotic bacteria daily, the other half consuming thesame amount of control cheese. The cheeses were eaten unheated after themorning and evening meals, medication and teeth wash. The probioticscheese contained 10⁷ cfu/g of L. rhamnosus LGG, 10⁷ cfu/g of L.rhamnosus LC705, and 10⁷ cfu/g of P. freudenreichii ssp. shermanii PJS.Thus the total amount of bacteria was 10⁹-10¹⁰ cfu/day.

Clinical mouth controls: Mouth controls were carried out and salivasamples taken at the old people's homes/nursing homes where the testpersons lived in. The clinical checks included recording filled andremoved teeth and those affected by caries (DMF). Also the condition ofthe periodontium was checked and classified according to the CPI index.Changes, if any, in the mucosal membrane of the mouth were recorded(including infections, discolorations, wounds, lichenoid lesions andulcers, hyperplasia, leucoplakia, erythroplakia, and other). Theclinical controls were carried out at the beginning of the study (0 wk)and at the end of it (16 wk).

Saliva samples: Saliva samples to determine yeasts, the rate of salivasecretion and buffer capacity were taken between 8 a.m. and 11 a.m.every morning. An attempt was made to take the sample always at the sametime from each person. The test persons were not allowed to eat or washtheir teeth for one hour before the taking of the sample. Saliva samplesfor yeast analysis were taken at the beginning of the study (0 wk), inthe middle of it (8 wk) and at the end of it (16 wk). The rate of salivasecretion and the buffer capacity were determined from the first sample(0 wk) and the last samples (16 wk).

Yeasts were determined from saliva by taking samples from the mucosalmembrane of the mouth with a cotton stick. The yeasts (mainly Candidaalbicans) were detected using the Dentocult CA cultivation method, inwhich the tubes are incubated in an incubator at 37° C. for 2 days,yeast growth being then determined semi-quantitatively on a scale of 0-3(0=no colonies, 1=1-20 cfu/slip, 2=21-50 cfu/slip, 3>50 cfu).

Saliva secretion rate was determined by measuring both resting salivaand stimulated saliva. The limit value for hyposalivation is consideredto be 0.1 ml/min of resting saliva; therefore resting saliva wascollected for 15 min (1.5 ml/15 min). Stimulated saliva was collectedfor 5 minutes (the hyposalivation limit being 3.5 ml/5 min).

Saliva buffer capacity is associated with the rate of secretion. Forthis reason buffer capacity was measured using the Dentobuff test. Thetest was carried out on stimulated saliva, because the buffer capacityof resting saliva is always poor.

At the end of the intervention period the amount of yeast formed theprimary response variable in the study. The differences in theoccurrence and the amounts of yeast between the groups were tested usingthe Chi Square test. In addition, the occurrence of yeast or the amountthereof in the baseline situation and demographic factors (such as age,gender, prostheses), if any, were taken into account using a logisticregression analysis. In this connection, the saliva secretion rate andbuffer capacity were also taken into account as explanatory factors.High saliva contents were analysed correspondingly. Changes in salivasecretion rate and the buffer capacity were described.

The results are shown in the following tables, in which A represents thegroup that ate the probiotics cheese, B being the control group. Thenumber of colonies has been presented as follows:

0=no colonies

1=1-20 cfu/slip

2=20-50 cfu/slip

3=more than 50 cfu/slip

Table 1 shows the precise yeast measurement results at the beginning ofthe study and after 8 and 16 weeks of intervention for persons from whomall the three measurements were obtained (in group A n=92, in group Bn=100). Table 2 shows classified yeast amounts at the beginning of thestudy and after 8 and 16 weeks of intervention for persons from whom allthe tree measurements were obtained (in group A n=92, in group B n=100).TABLE 1 Precise yeast results at the beginning of the study and after 8and 16 weeks of intervention A B n % n % Baseline 0 27 29.3 30 30.0 1 3740.2 42 42.0 2 17 18.5 14 14.0 3 11 12.0 14 14.0  8 wk 0 32 34.8 37 37.01 37 40.2 32 32.0 2 10 10.9 15 15.0 3 13 14.1 16 16.0 16 wk 0 35 38.0 3333.0 1 38 41.3 33 33.0 2 11 12.0 17 17.0 3 8 8.7 17 17.0

TABLE 2 Classified yeast contents at the beginning of the study andafter 8 and 16 weeks of intervention A B n % n % 0 vs. 1-4 Baseline 0 2729.3 30 30.0 1-4 65 70.7 70 70.0  8 wk 0 32 34.8 37 37.0 1-4 60 65.2 6363.0 16 wk 0 35 38.0 33 33.0 1-4 57 62.0 67 67.0 0-1 vs. 2-4 Baseline0-1 64 69.6 72 72.0 2-4 28 30.4 28 28.0  8 wk 0-1 69 75.0 69 69.0 2-4 2325.0 31 31.0 16 wk 0-1 73 79.3 66 66.0 2-4 19 20.7 34 34.0 0-2 vs. 3-4Baseline 0-2 81 88.0 86 86.0 3-4 11 12.0 14 14.0  8 wk 0-2 79 85.9 8484.0 3-4 13 14.1 16 16.0 16 wk 0-2 84 91.3 83 83.0 3-4 8 8.7 17 17.0

Groups A and B were compared to each other for yeast occurrence after 8and 16 weeks of intervention both directly and taking into accountinterfering factors, if any. These factors consist of age, gender, typeof living, number of diagnoses, amount of medication, BMI, saliva flowrate, buffer capacity, and prosthesis, if any. The interfering factorswere taken into account by using stepwise logistic regression. The groupand the baseline yeast situation are forced into a model (block 1) andthe interfering factors interfering on the list are taken into accountstepwise (block 2; significance criterion p being less than 0.15). Theresults are shown in the following tables, in which table 3 shows adirect comparison in the group of relatively high yeast occurrence, i.e.classes 2-4, table 4 takes into account the interfering factors, table 5shows a direct comparison in the group of high yeast occurrence, i.e.classes 3-4, and table 6 takes into account the interfering factors. Inthese tables abbreviation OR=odds ratio and abbreviation Cl forOR=confidence interval for odds ratio. TABLE 3 Relatively high yeastoccurrence (classes 2-4) B p-value OR 95% CI for OR a) after 8 weeksGroup A −0.469 0.178 0.625 0.316 to 1.239  Initial yeast 2-4 2.382<0.0001 10.825 5.450 to 21.500 Constant −1.520 <0.0001 b) after 16 weeksGroup A −0.946 0.014 0.388 0.182 to 0.826  Initial yeast 2-4 2.244<0.0001 9.4355 4.461 to 19.957 Constant −1.390 <0.0001

TABLE 4 Relatively high yeast occurrence (classes 2-4), with interferingfactors taken into account B p-value OR 95% CI for OR Step 1 Group A−1.229 0.0082 0.293 0.118 to 0.728  Initial yeast 2-4 1.911 <0.00016.757 2.875 to 15.884 Buffer capacity 0.0002 Buffer capacity −2.318<0.0001 0.098 0.032 to 0.299  (H) Buffer capacity −1.614 0.0113 0.1990.057 to 0.694  (M) Constant 0.481 0.3896 1.617 Step 2 Group A −1.3780.0043 0.252 0.098 to 0.649  Initial yeast 2-4 1.845 <0.0001 6.325 2.636to 15.178 Buffer capacity 0.0003 Buffer capacity −2.388 <0.0001 0.0920.029 to 0.290  (H) Buffer capacity −1.588 0.0152 0.204 0.057 to 0.737 (M) Prosthesis 1.117 0.0390 3.057 1.058 to 8.831  (status 1) Constant−0.246 0.7165 0.782

TABLE 5 High yeast occurrence (classes 3-4) B p-value OR 95% CI for ORa) after 8 weeks Group A 0.029 0.956 1.029 0.373 to 2.837  Initial yeast3-4 4.022 <0.0001 55.8  18.7 to 166.8  Constant −2.782 <0.0001 b) after16 weeks Group A −0.824 0.098 0.439 0.165 to 1.164  Initial yeast 3-42.420 <0.0001 11.244 4.186 to 30.208 Constant −2.142 <0.0001

TABLE 6 High yeast occurrence (classes 3-4), with interfering factorstaken into account B p-value OR 95% CI for OR Step 1 Group A −1.0670.0657 0.344 0.110 to 1.072  Initial yeast 3-4 2.221 0.0003 9.216 2.798to 30.359 Buffer capacity 0.0370 Buffer capacity −1.629 0.0142 0.1960.053 to 0.721  (H) Buffer capacity −0.551 0.4525 0.576 0.137 to 2.428 (M) Constant 0.982 0.1136 0.375 Step 2 Group A −1.213 0.0437 0.297 0.091to 0.966  Initial yeast 3-4 2.300 0.0002 9.974 2.979 to 33.396 Buffercapacity 0.0238 Buffer capacity −1.807 0.0080 0.164 0.043 to 0.624  (H)Buffer capacity −0.726 0.3339 0.484 0.111 to 2.109  (M) Generation of-0.956 0.0991 0.385 0.124 to 1.197  women Constant −0.154 0.8452 0.857Step 3 Group A −1.475 0.0222 0.229 0.065 to 0.810  Initial yeast 3-42.213 0.0004 9.145 2.704 to 30.924 Buffer capacity 0.0256 Buffercapacity −1.846 0.0086 0.158 0.040 to 0.625  (H) Buffer capacity −0.7810.3151 0.458 0.100 to 2.101  (M) Generation of −1.371 0.0305 0.254 0.073to 0.879  women Prosthesis 1.274 0.1003 3.576 0.782 to 16.350 (status 1)Constant −0.713 0.4344 0.490

The results show that ageing, use of prostheses and reduced salivationare clearly associated with increased amount of yeast. Lower initialyeast amounts, in turn, were discovered with test persons who reportedthat they had regularly used products containing lactic acid bacteriabefore the study.

When examining the yeast results alone, without taking into accountinterfering factors, it can be seen that yeast amounts decreased more inthe probiotics group than in the control group. Irrespective of whetherthe focus is on studying yeast occurrence (1-4), relatively high yeastoccurrence (2-4), or high yeast occurrence (3-4), the results show thatthe proportion of those belonging to the probiotics group decreases inall of these groups as the intervention proceeds. The proportion ofthose belonging to the control group, in turn, does not change equallyclearly. On the contrary, the proportion of those belonging to thegroups of higher yeast occurrence seems even to increase in the controlgroup.

When interfering factors are taken into account, it is observed thatprobiotics intervention produces a statistically significant reductionin the occurrence of both relatively high (2-4) and high (3-4) yeastamounts, as compared with the control group.

The results thus show that by using cheese containing the aboveprobiotics, it was possible to reduce yeast amounts significantly. Thepresent invention is thus useful for inhibiting yeast in accordance withthe objectives of the invention.

1. Use of the microbes Lactobacillus rhamnosus LGG, ATCC 53103,Lactobacillus rhamnosus LC705, DSM 7061, and Propionibacteriumfreudenreichii ssp. shermanii PJS, DSM 7067 for preparing a product forinhibiting yeast.
 2. Use of microbes according to claim 1, wherein bypreparing a product of the food industry or the pharmaceutical industry,a health promoting product, or natural products.
 3. Use of microbesaccording to claim 1, wherein by preparing a food product.
 4. Use ofmicrobes according to claim 3, wherein by preparing a dairy product,preferably cheese.
 5. Use of microbes according to claim 1, wherein inthat also other, conventional (starter) bacteria are used in thepreparation.
 6. Use of microbes according to claim 1, wherein bypreparing a unit dosage form containing the microbes.
 7. Use of microbesaccording to claim 6, wherein in that the dosage form is a preparationfor oral consumption.
 8. Use of microbes according to claim 7, whereinin that the dosage form is a capsule or a tablet containing xylitol, inaddition to the microbes.
 9. A method for inhibiting the growth ofyeasts and for relieving yeast-related symptoms in animals or humans,the method comprising: administering the microbes Lactobacillusrhamnosus LGG, ATOC 53103, Lactobacillus casei ssp. rhamnosus LC705, DSM7061, and Propionibacterium freudenreichii ssp. shermanii PJS, DSM 7061to an individual in the need thereof in an amount sufficient to producethe desired effect.